Dose and localization of botulinum toxins in skin and muscle

ABSTRACT

A novel dosing regimen for the administration of botulinum toxin based on the pattern, quantity, and location of neuromuscular junctions in the target tissue. Because the number of neuromuscular junctions in a target tissue remains generally stable throughout life and because the pharmacological effect of botulinum toxin is localized at the neuromuscular junction, dosing efficacy is unaffected by muscle mass, age of the patient, or body weight.

RELATED APPLICATIONS

This is a utility patent application claiming priority from U.S.Provisional Application No. 61/136,908, filed Oct. 14, 2008.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None

REFERENCE TO SEQUENCE LISTING, A TABLE OR A COMPUTER PROGRAM LISTINGCOMPACT DISC APPENDIX

None.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to a dosing protocol for the administration ofbotulinum toxin that maximizes efficacy and specificity while minimizingthe likelihood of overdosing and undesirable side effects of botulinumtoxin treatment.

2. Background of the Invention

Botulinum toxins, in particular botulinum toxin type A, have been usedin the treatment of a number of neuromuscular disorders and conditionsinvolving muscular spasm as well as in cosmetic procedures; for example,strabismus, blepharospasm, spasmodic torticollis (cervical dystonia),oromandibular dystonia and spasmodic dysphonia (laryngeal dystonia). Thetoxin binds rapidly and strongly to presynaptic cholinergic nerveterminals and inhibits the exocytosis of acetylcholine by decreasing thefrequency of acetylcholine release thereby reducing or eliminating theactivation of postsynaptic muscles, nerves, or effector tissues. Thisresults in local paralysis and hence relaxation of the muscle afflictedby spasm.

The term botulinum toxin as used herein is a generic term embracing thefamily of toxins produced by the anaerobic bacterium Clostridiumbotulinum and, to date, seven immunologically distinct toxins have beenidentified. These have been given the designations A, B, C, D, E, F andG. For further information concerning the properties of the variousbotulinum toxins, reference is made to the article by Jankovic & Brin,The New England Journal of Medicine, pp 1186-1194, No 17, 1991 and tothe review by Charles L Hatheway, Chapter 1 of the book entitledBotulinum Neurotoxin and Tetanus Toxin Ed. L. L. Simpson, published byAcademic Press Inc. of San Diego Calif. 1989, the disclosures in whichare incorporated herein by reference.

The neurotoxic component of botulinum toxin has a molecular weight ofabout 150 kilodaltons and is believed to comprise a short polypeptidechain of about 50 kD which is considered to be responsible for the toxicproperties of the toxin, and a larger polypeptide chain of about 100 kDwhich is believed to be necessary to enable the toxin to penetrate thenerve. The “short” and “long” chains are linked together by means ofdisulphide bridges.

Intramuscular injections of botulinum toxin A are generally used tobalance muscle forces across joints, to diminish or decrease painfulspasticity, to decrease deforming forces through selective motorparalysis, to diminish neuropathic and nociceptive pain, to diminishdystonic contractures, to decrease muscle deformation after injury orsurgery, and to diminish sweating. The target organelles contain solubleNSF attachment receptor (SNARE) proteins and neurotransmitter-containingvesicles which require these SNARE proteins for fusion of the vesicle tothe cell membrane and release of neurotransmitter. Targets includeneuromuscular junctions, sweat glands, vascular beds and nociceptors.

Therapeutic use of these toxins represents a somewhat uniquepharmacokinetic profile. In order for toxin to produce its desiredaction, it must not only be delivered to the target tissue, e.g. muscle(usually by direct injection), but it must also bind to terminalportions of nerves innervating the target tissue (i.e. the neuromuscularjunction), and be transported across the presynaptic terminal membraneinto the intracellular domain where the active molecule is cleaved fromthe binding portion of the divalent complex. Then the active moleculemust bind irreversibly and enzymatically inactivate molecules in thenerve terminal specific for neurochemical transmission. Thus the toxinmolecules are not delivered systemically to distribute throughout thebody. The ultimate target is not a specific muscle or organ but rathermolecules located in specific nerves which innervate the target tissuewithin an anatomically defined region of the target tissue or muscle.For example, within skeletal muscle fibers, nerves do not uniformlydistribute through the muscle but rather the terminals of the nerves arerestricted to a certain region of the muscle. In the case of musclefibers, prior research has shown that different muscles have differentnumbers of neuromuscular junctions and the total number of theseneuromuscular junctions is not dependent on the mass or volume of themuscle or the individual but rather on other factors such as thefunction of the muscle fibers.

Current recommendations and dosing regimens are empirical and utilizedosage based upon bodyweight in, for example, the management of cerebralpalsy and in orthopaedic uses. With specific regard to its use inchildren, the use of botulinum toxin in the management of cerebral palsyand in orthopaedic usage is based on the size and weight of the growingchild, rather than age, to insure safety since overall toxicity data wasbased upon units per kilogram of body weight in primates. U.S. Pat. No.6,395,277 issued 28 May 2002 shows a dosing regimen for the treatment ofcerebral palsy, noting that dosing should occur “preferably . . . in theregion of the neuromuscular junction” according to “the number of musclegroups requiring treatment, the age and size of the patient.” Similardosing regimens base relative dosages upon the size of muscle.

Historically, dosage recommendations for administration of botulinumtoxin has been an imprecise science. Recommendations have been made onthe basis of body weight, body surface area, size or volume occupied bya specific muscle, etc. The overreaching goal for each of thesetherapeutic or cosmetic uses of botulinum toxins is that the toxin beadministered in a dosage and volume appropriate to achieving the desiredresponse while remaining localized within the desired specific region ofinjection. Because the ultimate site of toxin action is nerve junctionswithin certain regions of the target tissue, over- and under-dosingremains a significant challenge. Administration of too high an absolutedose (total number of toxin molecules relative to the total number ofneuromuscular junction targets) or too high a volume of injection mightproduce adverse reactions related to diffusion of the toxin. Diffusionof the toxin into undesired areas could produce inappropriate paralysisor pathophysiological responses. Too high a dose will produce thedesired effect of tissue paralysis but also result in toxin distributionto non targeted tissues thereby causing an unintended loss ofphysiological function in these regions. Additionally, delivery ofsupraoptimal toxin doses presents an undesired immunological challengewhich may cause reduced effectiveness on subsequent administrations ofthe toxin. When a large volume of toxin is delivered, it is likely thattoxin molecules will diffuse to distant targets resulting in thedilution of the effect of the toxin at the desired target andinappropriately exposing other regions to the toxin. In a large volumedosing scenario, a higher overall dose of toxin would be required at alater time to overcome the dilution effect thus increasing the exposureof other tissues. In these cases where inappropriate doses or volumesare used, not only may patient safety be compromised but the cost of theprocedure is increased due to wasted toxin or treatment of unanticipatedpharmacological outcomes.

Perhaps the most obvious examples of this inappropriate dosage aredelivery of toxin based on body weight to individuals who are at theextremes of weight distribution curves. The toxin acts at theneuromuscular junction and the quantity of the aforementioned junctionsdoes not change proportionately with changes in body mass. Hence, inthese cases, individuals with high and low body mass would receiveinappropriately high or low doses, respectively.

Various recommendations have demonstrated clinical usefulness but failto address that 1) the toxin acts at the neuromuscular junction, and 2)the number of neuromuscular junctions varies from muscle to muscle, and3) the number of neuromuscular junctions tends not to vary as a personages. Neuromuscular junctions for individual muscles are not directlyproportional to muscle mass or volume. Rather, the distribution ofneuromuscular junctions varies from muscle to muscle and the number ofneuromuscular junctions is affected minimally by age and total bodyweight. The existing dosage recommendations are clinically efficaciousin 50 to 70 percent of patients, namely large toddlers and adolescents,but may underdose infants and small toddlers and overdose heavychildren, teenagers, and adults. What is needed are more precise dosingmethods to delineate optimal number of units, volume, and injectionsites for individual muscles, thereby improving efficacy, minimizingprotein antigen load and subsequent antibody formation, and decreasingcosts.

SUMMARY OF THE INVENTION

The present invention is a novel dosing method for botulinum toxin basedon the number and distribution of neuromuscular junctions in the targetmuscle. It includes determining the mass of the target muscle,determining the distribution and location of neuromuscular junctions inthat muscle, and injecting an appropriate therapeutic dose of botulinumtoxin in the vicinity of and according to the quantity of neuromuscularjunctions in the muscle. A dosing regimen based on the quantity ofneuromuscular junctions in the aforementioned tissue ensures efficacy,while minimizing possible side effects and minimizing cost by ensuringthat only that amount of toxin necessary to achieve the desired effectis used.

It is an object of this invention to provide a safe dosing method forbotulinum toxins;

It is another object of this invention to provide an efficacious methodfor dosing botulinum toxins;

It is still another object of this invention to provide a minimallyinvasive means of dosing botulinum toxins;

It is yet another object of this invention to provide a cost effectivedosing method for botulinum toxins; and,

It is an object of this invention to provide a simple, easily compliedwith dosing method for the use of botulinum toxins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a rat gastrocnemius muscle;

FIG. 2 graphs the dose response recovery of botulinum toxin at dosagesof 1 U/kg, 3 U/kg and 6 U/kg;

FIG. 3 graphs the effect of different volumes of toxin injection;

FIG. 4 shows a unipennate muscle with a single transverse band ofneuromuscular junctions;

FIG. 5 shows a unipennate gracilis muscle with two transverse bands ofneuromuscular junctions;

FIG. 6 shows a bipennate converging biceps brachii muscle havingneuromuscular junctions located in an inverted “U” shape;

FIG. 7 shows a rectus femoris muscle with two bands of neuromuscularjunctions running along its length; and,

FIG. 8 shows a deltoid muscle with an irregular pattern of neuromuscularjunctions.

These and other objects, advantages, and novel features of the presentinvention will become apparent when considered with the teachingscontained in the detailed disclosure along with the accompanyingdrawings.

DESCRIPTION OF THE INVENTION

While the invention is described in connection with certain preferredembodiments, it is not intended that the present invention be solimited. On the contrary, it is intended to cover all alternatives,modifications, and equivalent arrangements as may be included within thespirit and scope of the invention as defined by the appended claims.

The invention is a novel dosing method for botulinum toxin based on thequantity and distribution of neuromuscular junctions in a target tissue.Previous recommendations for dosing were based on, for example, bodymass and/or age. In the present invention, therapeutic dosing is basedon 1) the quantity and distribution of neuromuscular junctions and 2)the volume of liquid or other carrier material in which that dose isdelivered to the target tissue. This results in decreased incidences ofunder or over-dosing, minimized direct costs of administrating thesubstance due to the more efficient use of the toxin itself, andminimized indirect costs resulting from the medical costs avoided byeliminating the likelihood of anaphylaxis and immuno-challenge resultingfrom too high a dose. Ideal dose in units is therefore based upon thenumber of NSF attachment receptor (SNARE) containing organelles (i.e.,neuromuscular junctions to be blocked); the volume or concentrationcalculated from the muscle mass; and the number of injection sites isdictated by the length and width of the target tissue.

As shown in the Dose Response Recovery Graph of FIG. 2, because itsefficacy is dependant on the quantity of neuromuscular junctions in thetarget tissue, dosing of botulinum toxin exhibits clear maximum dosingbehavior beyond which an increased dose shows no appreciable change ineffect. The neuromuscular junctions of the target tissue are saturatedsuch that additional availability of toxin produces no additionaleffect. The graph of FIG. 3, however, shows clearly that propertitration of the dose is important. While suboptimal amounts of toxinobviously produce lower degrees of relaxation, supraoptimal dosesproduce similarly reduced results. It is believed that the reducedresults occur because toxin molecules diffuse away from the target siteresulting in the dilution of the effect of the toxin at the desiredtarget and inappropriately expose other regions to the toxin. Hencedetermination of the most efficacious dosage for a target muscle iscritical.

Botulinum toxin A is produced by Allergan Pharmaceuticals as BOTOX® andby Ipsen Pharmaceuticals, Ltd. as DYSPORT®. Each vial of BOTOX® contains100 units of C. botulinum type A neurotoxin complex, 0.5 mg of humanalbumin, and 0.9 mg of sodium chloride as a vacuum-dried frozen powderthat requires reconstitution. One unit of BOTOX® is equal to the medianintraperitoneal lethal dose (LD50) in Swiss-Webster mice weighing 18 to20 g. The LD50 for BOTOX® has been calculated in primates at 39 to 56units/kg body wt. However, the exact lethal dose in humans is unknown.The calculated human LD50 of 59 units is based on an extrapolation ofdata. DYSPORT® clostridium botulinum type A toxin-hemogglutinin complexis available in 500-unit vials. DYSPORT® units of activity equal 1 mouseLD50 based on their specific assay technique and is sometimes referredto in nanograms, with 1 nanogram equal to 40 units. In the UnitedKingdom and many other countries, it is approved and labeled formultiple indications, including spasticity of the arm in patientsfollowing stroke, dynamic equinus foot deformity due to spasticity inambulant pediatric cerebral palsy patients, two years of age or older,spasmodic torticollis, blephorospasm, and hemifacial spasm. With regardto cerebral palsy, DYSPORT® dosing is recommended as “30 units/kg bodyweight divided between both calf muscles.”.

Existing clinical data supports that BOTOX® and DYSPORT® potencies aredifferent; one BOTOX® unit is equal to 2 to 4 DYSPORT® units. Units arenot interchangeable between companies or toxin types using packageguidelines and suggested dilution tables.

Both BOTOX® and DYSPORT® are reconstituted in injectable physiologicsaline prior to intramuscular injection. Both the volume of fluid andnumber of units of drug must be considered when preparing the toxin forinjection. Dosage is defined in absolute terms, based on the number ofunits per target muscle diluted to volume based on the size of thestructure to be injected and quantity and distribution of neuromuscularjunctions. The number of units to be injected is calculated by thequantity of neuromuscular junctions to be neutralized, and the volume isdetermined by the mass of the target muscle, and the number of injectionsites by the anatomic distribution of the neuromuscular junctions. Oncethe appropriate number of active toxin molecules (units) for a givenmuscle is determined, the dose in units remains constant and the volumeand number of injection sites is adjusted based upon growth and anatomy.For example, there are an estimated pikamole of active toxin moleculesin 100 units of BOTOX® and an estimated 250,000 neuromuscular junctionsin the human biceps brachii. Hence, there are sufficient active toxinmolecules to block effectively all neuromuscular junctions of the“target” muscle. The toxin is thereafter injected within the muscle orskin as close to the neuromuscular junctions (or other SNARE-containingorganelle) as possible using ultrasonography to localize their position.

Visualization of extremity and trunk muscles is performed reliably usinglinear probe ultrasonography with a frequency of 5-12 Mhz. For injectionlocalization, linear beam applications better define and delineate theanatomic relationships between muscles, tendons or bones. Higherfrequencies are recommended for the localization of the superficialmuscles or layers, while lower frequencies may be used for deepstructures. The muscles are covered by the epimysium which is theconnective tissue that surrounds the entire muscle. The epimysiumextends into the muscle to become the perimysium, which divides thefascicle into muscle fibers. The perimysium and the muscular fasciclescan be identified because the muscular bundles are hypoechoic (lessbright) while the epimysium and perimysium appear as hyperechoicstructures. On longitudinal scanning, the fascia is depicted as afibrillar hyperechoic sheath surrounding the muscle.

There are approximately 250,000 neuromuscular junction in the humanbiceps brachii muscle. Other human extremity muscles (e.g., the lateraland medial head of the gastrocnemius) have a similar neuromuscularjunction density. The total dosage of botulinum toxin (i.e., theabsolute number of toxin molecules administered) is given based on themass of the muscle rather than on the body weight of the individual andinjected within 3.0 cm of the area of the muscle containing theneuromuscular junctions (based on ultrasound localization). Thus, formuscles like the soleus where junctions are distributed along the lengthof muscle fibers, toxin is delivered in multiple locations following thefull length of the muscle. In contrast, for muscles like the bicepsbrachii or medial and lateral head of the gastrocnemius the injectionpattern is an inverted U shape following the distribution of theneuromuscular junctions. For example, where 75U is sufficient to produceblockade of the neuromuscular junctions in the lateral gastrocnemius,the biceps brachii has a mass 22% larger than the lateral gastrocnemius,therefore requiring 91U for efficacy. These absolute amounts are thendiluted relative to increasing mass and injected adjacent theneuromuscular junctions in the target muscle.

Table 1 shows muscles relative to the lateral gastrocnemius andrecommended dosages.

TABLE 1 Range of Body wt. (kg) 0-5 5-10 10-20 20-40 Concentration (U/ml)100 50 25 12.5 Total U of Botox mass relative 75 75 75 to lateral 75 2X3X 4-5 X muscle gastrocnemius m. ml per muscle ml per muscle ml permuscle ml per muscle lateral gastrocnemius 1.00 0.8 1.5 2.3 3.4 medialgastroc 1.48 1.1 2.2 3.3 5.0 tibialis posterior 0.84 0.6 1.3 1.9 2.8tibialis anterior 0.66 0.5 1.0 1.5 2.2 soleus 2.63 2.0 3.9 5.9 8.9 FHL0.44 0.3 0.7 1.0 1.5 Sartorius 0.56 0.4 0.8 1.3 1.9 Semimembranosus 0.980.7 1.5 2.2 3.3 Semitendinosus 0.70 0.5 1.1 1.6 2.4 Gracilis 0.32 0.20.5 0.7 1.1 Pronator Teres 0.23 0.2 0.3 0.5 0.8 Biceps 1.22 0.9 1.8 2.74.1 Brachioradialis 0.39 0.3 0.6 0.9 1.3 Pronator Quadratus 0.07 0.1 0.10.2 0.2 Supinator 0.21 0.2 0.3 0.5 0.7 FCU 0.14 0.1 0.2 0.3 0.5 FCR 0.100.1 0.2 0.2 0.3 FDS 0.12 0.1 0.2 0.3 0.4 FDP 0.12 0.1 0.2 0.3 0.4 ECRB0.13 0.1 0.2 0.3 0.4 Subscapularis 1.02 0.8 1.5 2.3 3.4 Teres Minor 0.160.1 0.2 0.4 0.5 Infraspinatus 0.76 0.6 1.1 1.7 2.6 Supraspinatus 0.310.2 0.5 0.7 1.0

Table 1 provides a multiplication factor by which the appropriate dosagefor other muscles may be determined For example, the soleus muscle has amass approximately 2.63 times greater than the lateral gastrocnemius.Where 75U of toxin is efficacious for relaxation of the lateralgastrocnemius, and approximately 0.8 ml of a 100 U/ml concentration oftoxin is administered in a patient with a body weight of up to 5 kg,approximately 2.0 ml (i.e., 2.63 times 0.8 ml) is efficacious forrelaxation of the soleus. Note that as body weight doubles to 10 kg, 20kg, and 40 kg, toxin is diluted accordingly but injected in sufficientvolume such that the absolute amount of botulinum toxin administeredremains the same regardless of muscle size. Increasing muscle mass doesnot require additional toxin because the number of neuromuscularjunctions does not change.

FIG. 4 shows a unipennate muscle 10 with a single transverse band ofneuromuscular junctions 50. Intramuscular injection of toxin is mostefficacious when delivered within 3.0 cm of this band. Similarly FIG. 5shows unipennate gracilis muscle 11 with two transverse bands ofneuromuscular junctions 50. FIG. 6 shows a bipennate converging bicepsbrachii muscle 12 having neuromuscular junctions 50 located in aninverted “U” shape. FIG. 7 shows a rectus femoris muscle 13 with twobands of neuromuscular junctions 50 running along its length, and FIG. 8shows a deltoid muscle 14 with an irregular pattern of neuromuscularjunctions 50.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.However, the invention should not be construed as limited to theparticular embodiments which have been described above. Instead, theembodiments described here should be regarded as illustrative ratherthan restrictive. Variations and changes may be made by others withoutdeparting from the scope of the present invention as defined by thefollowing claims:

What is claimed is:
 1. A method for dosing botulinum toxin in a livinghuman muscle of a patient comprising the steps of: a) determining themass of a target muscle relative to the mass of a lateral gastrocnemiusmuscle of the patient; b) determining the distribution and location ofneuromuscular junctions (NMJ) in said target muscle as mediated byultrasound localization; and, c) injecting a dosing regimen comprising aplurality of volumetrically optimized doses of botulinum toxin within adistance of about 3.0 cm of said neuromuscular junctions relative to thequantity of said neuromuscular junctions in said target muscle and theratio of said mass of said lateral gastrocnemius muscle with the mass ofsaid target muscle with the number of doses being dictated by the lengthand width of the target muscle.
 2. The method of claim 1 wherein saidtarget muscle is a lateral gastrocnemius muscle and a dosage of about 75units of botulinum toxin is injected, each botulinum toxin unit having amedian intraperitoneal lethal dose in Swiss-Webster mice weighing 18 to20 grams, said dosage being administered in an inverted U-shaped patternadjacent said lateral gastrocnemius neuromuscular junctions.
 3. Themethod of claim 1 wherein said target muscle is a medial gastrocnemiusmuscle and is about 1.48 times the mass of a lateral gastrocnemiusmuscle and a dosage of about 111 units of botulinum toxin is injected,each botulinum toxin unit having a median intraperitoneal lethal dose inSwiss-Webster mice weighing 18 to 20 grams, said dosage beingadministered in a substantially inverted U-shaped pattern adjacent saidmedical gastrocnemius neuromuscular junctions.
 4. The method of claim 1wherein said target muscle is a semitendinosus muscle and is about 0.70times the mass of a lateral gastrocnemius muscle and a dosage of about53 units of botulinum toxin is injected, each botulinum toxin unithaving a median intraperitoneal lethal dose in Swiss-Webster miceweighing 18 to 20 grams, said dosage being administered about atransverse midline adjacent said semitendinosus neuromuscular junctions.5. The method of claim 1 wherein said target muscle is a brachioradialismuscle and is about 0.39 times the mass of said lateral gastrocnemiusmuscle and a dosage of about 29 units of botulinum toxin is injected,each botulinum toxin unit having a median intraperitoneal lethal dose inSwiss-Webster mice weighing 18 to 20 grams, said dosage beingadministered about a transverse midline adjacent said brachioradialisneuromuscular junctions.
 6. The method of claim 1 wherein said targetmuscle is a gracilis muscle and is about 0.32 times the mass of saidlateral gastrocnemius and a dosage of about 24 units of botulinum toxinis injected, each botulinum toxin unit having a median intraperitoneallethal dose in Swiss-Webster mice weighing 18 to 20 grams, said dosagebeing administered about two transverse lines adjacent said gracileaneuromuscular junctions.
 7. The method of claim 1 wherein said targetmuscle is a biceps brachii muscle and is about 1.22 times the mass ofsaid lateral gastrocnemius muscle and a dosage of about 92 units ofbotulinum toxin is injected, each botulinum toxin unit having a medianintraperitoneal lethal dose in Swiss-Webster mice weighing 18 to 20grams, said dosage being administered in a substantially invertedU-shaped pattern adjacent said biceps brachii neuromuscular junctions.8. The method of claim 1 wherein said target muscle is a soleus muscleand is about 2.63 times the mass of said lateral gastrocnemius muscleand a dosage of about 197 units of botulinum toxin is injected, eachbotulinum toxin unit having a median intraperitoneal lethal dose inSwiss-Webster mice weighing 18 to 20 grams, said dosage beingadministered along the length of the muscle fibers adjacent said soleusneuromuscular junctions.